Abstract Abstract Metallothioneins, which are a kind of widely distributed and low molecular weight cysteine-rich proteins, were found to play an important role in heavy metal tolerance. In this study, a cDNA fragment corresponding to the type Ⅱ metallothionein (MT2) coding region (GenBank No. KY643823) was isolated from tartary buckwheat (Fagopyrum tataricum) leaf. FtMT2 encoded a deduced peptide of 78 aa residues with a calculated molecular mass of 7.87 kD and contained the cysteine-rich domains with the presence of C-C, C-X-C, and C-X-X-C (X was other amino acids other than cysteine) motifs at amino-terminus and C-X-C motifs at carboxy-terminus. Sequence and homology analysis showed that the FtMT2 protein sequence shared high homology with other plant type 2 MT-like proteins. In order to clarify the regulatory mechanism of FtMT2 in response to Cu2+ stress, 2.1 kb upstream sequence of FtMT2 was isolated by genome walking. The 2.1 kb fragment and its 5′ flanking deletions were tested for promoter activity using the dual luciferase reporter system and transient transformation in mesophyll protoplasts of F. tararicum. The promoter activities of all the fragments were up-regulated, and the full length promoter showed strongest inducibility in response to Cu2+ treatment. The role of FtMT2 protein in protecting against Cu2+ toxicity was demonstrated in pGEX4T-1-FtMT2-beared Escherichia coli, which could obviously increase the host E. coli cell's tolerance. Functional characterization of upstream region of FtMT2 provides the regulatory mechanism for establishing the method for copper contamination monitoring and offers an underlying basis for further revealing the molecular mechanisms of heavy metal resistance of Tartary buckwheat.
Keywords Tartary buckwheat, Metallothioneins (MTs), Promoter, Heavy metal tolerance
GU Peng,YU Yuan,CHEN Shou, et al. Cloning of MT2 from Tartary Buckwheat (Fagopyrum tataricum) and Its Response to Cu2+ Stress[J]. , 2017, 25(6): 874-883.
Ariyasu, S., A. Onoda, R. Sakamoto, et al. 2009. Conjugation of Au11 cluster with Cys-rich peptides containing the [small alpha]-domain of metallothionein[J]. Dalton Transactions(19): 3742-3747. Balasundaram, U., G. Venkataraman, S. George, et al. 2014. Metallothioneins from a Hyperaccumulating Plant Prosopis juliflora Show Difference in Heavy Metal Accumulation in Transgenic Tobacco[J]. International Journal of Agriculture, Environment and Biotechnology 7(2): 241. Berta, M., A. Giovannelli, E. Potenza, et al. 2009. Type 3 metallothioneins respond to water deficit in leaf and in the cambial zone of white poplar (Populus alba)[J]. J Plant Physiol 166(5): 521-530. Cai, L.-M., Z.-C. Xu, J.-Y. Qi, et al. 2015. Assessment of exposure to heavy metals and health risks among residents near Tonglushan mine in Hubei, China[J]. Chemosphere 127: 127-135. Chang, T., X. Liu, H. Xu, et al. 2004. A metallothionein-like gene htMT2 strongly expressed in internodes and nodes of Helianthus tuberosus and effects of metal ion treatment on its expression[J]. Planta 218(3): 449-455. Chaturvedi, A. K., A. Mishra, V. Tiwari, et al. 2012. Cloning and transcript analysis of type 2 metallothionein gene (SbMT-2) from extreme halophyte Salicornia brachiata and its heterologous expression in E. coli[J]. Gene 499(2): 280-287. Dundar, E., G. D. Sonmez,T. Unver. 2015. Isolation, molecular characterization and functional analysis of OeMT2, an olive metallothionein with a bioremediation potential[J]. Mol Genet Genomics 290(1): 187-199. Foley, R.,K. Singh. 1994. Isolation of a Vicia faba metallothionein-like gene: expression in foliar trichomes[J]. Plant Molecular Biology 26(1): 435-444. Freisinger, E. 2009. Metallothioneins in plants[J]. Met Ions Life Sci 5: 107-153. Gu, C. S., L. Q. Liu, Y. H. Zhao, et al. 2014. Overexpression of Iris. lactea var. chinensis metallothionein llMT2a enhances cadmium tolerance in Arabidopsis thaliana[J]. Ecotoxicol Environ Saf 105: 22-28. Gu, L., Z. Han, L. Zhang, et al. 2013. Functional analysis of the 5' regulatory region of the maize GALACTINOL SYNTHASE2 gene[J]. Plant Sci 213: 38-45. Guo, J., L. Xu, Y. Su, et al. 2013. ScMT2-1-3, a metallothionein gene of sugarcane, plays an important role in the regulation of heavy metal tolerance/accumulation[J]. Biomed Res Int 2013: 904769. Kim, Y. O., Y. G. Lee, D. H. Patel, et al. 2012. Zn tolerance of novel Colocasia esculenta metallothionein and its domains in Escherichia coli and tobacco[J]. J Plant Res 125(6): 793-804. Li, Y., Y. Y. Chen, S. G. Yang, et al. 2015. Cloning and characterization of HbMT2a, a metallothionein gene from Hevea brasiliensis Muell. Arg differently responds to abiotic stress and heavy metals[J]. Biochem Biophys Res Commun 461(1): 95-101. Liu, J., X. Shi, M. Qian, et al. 2015. Copper-induced hydrogen peroxide upregulation of a metallothionein gene, OsMT2c, from Oryza sativa L. confers copper tolerance in Arabidopsis thaliana[J]. J Hazard Mater 294: 99-108. Mir, G., J. Domenech, G. Huguet, et al. 2004. A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress[J]. J Exp Bot 55(408): 2483-2493. Mudalkar, S., R. Golla, D. Sengupta, et al. 2014. Molecular cloning and characterisation of metallothionein type 2a gene from Jatropha curcas L., a promising biofuel plant[J]. Mol Biol Rep 41(1): 113-124. Qi, X., Y. Zhang,T. Chai. 2007. Characterization of a novel plant promoter specifically induced by heavy metal and identification of the promoter regions conferring heavy metal responsiveness[J]. Plant Physiol 143(1): 50-59. Rai, M., C. He,R. Wu. 2009. Comparative functional analysis of three abiotic stress-inducible promoters in transgenic rice[J]. Transgenic Res 18(5): 787-799. Ryvolova, M., S. Krizkova, V. Adam, et al. 2011. Analytical methods for metallothionein detection[J]. Current Analytical Chemistry 7(3): 243-261. Sekhar, K., B. Priyanka, V. D. Reddy, et al. 2011. Metallothionein 1 (CcMT1) of pigeonpea (Cajanus cajan, L.) confers enhanced tolerance to copper and cadmium in Escherichia coli and Arabidopsis thaliana[J]. Environmental and Experimental Botany 72(2): 131-139. Wu, Y., X. Lu, S. Zhuang, et al. 2016. Contamination Characteristics and Assessment of Manganese, Zinc, Chrome, Lead, Copper and Nickel in Bus Station Dusts of Xifeng, Northwest China[J]. Xia, Y., Y. Lv, Y. Yuan, et al. 2012. Cloning and characterization of a type 1 metallothionein gene from the copper-tolerant plant Elsholtzia haichowensis[J]. Acta Physiologiae Plantarum 34(5): 1819-1826. Zhang, J., M. Zhang, S. Tian, et al. 2014. Metallothionein 2 (SaMT2) from Sedum alfredⅡ Hance confers increased Cd tolerance and accumulation in yeast and tobacco[J]. PloS one 9(7): e102750. Zhang, M., T. Takano, S. Liu, et al. 2014. Abiotic stress response in yeast and metal-binding ability of a type 2 metallothionein-like protein (PutMT2) from Puccinellia tenuiflora[J]. Molecular Biology Reports 41(9): 5839-5849. Zhang, Y.-W., N. F. Y. Tam,Y. S. Wong. 2004. Cloning and characterization of type 2 metallothionein-like gene from a wetland plant, Typha latifolia[J]. Plant Science 167(4): 869-877. 张莉,李志西. 2009. 传统荞麦制品保健功能特性研究[J]. 中国粮油学报 24(3): 53-57.( Zhang L, Li Z-X. Functional Characteristics of Traditional Buckwheat Products. Journal of the Chinese Cereals and Oils Association, 2009. 24(3): p. 53-57.) 张钟仁,陈鹏. 2016. 苦荞叶肉细胞原生质体的分离纯化及瞬时转化[J]. 西北植物学报 36(1): 183-189.( Zhang Z-R, Chen P. Isolation, Purification and Transient Transformation of Mesophyll Protoplast in Tartary Buckwheat. Acta Botanica Boreali-Occidentalia Sinica, 2016. 36(1): p. 183-189.)
